(1) Technical Field
The present invention relates to a dark-field laser-scattering microscope for analyzing single macromolecules. More specifically, the present invention relates to a microscope with a variable wavelength, movable laser with well-defined direction for dark-field light-scattering of macromolecules in-vivo to reveal characteristics such as size, shape, molecular weight, and refractive index. The microscope is capable of constructing a three-dimensional image of a sample, with the use of a computer, without separating the sample from the in-vivo environment or contaminating the sample with fluorescent dyes.
(2) Background
There is great interest in analyzing single macromolecules with sizes well below 1 micron without having to separate them from the in-vivo environment. For example, certain vesicles, viruses, micro crystals, polymers and wax formations would be ideal candidates for analysis in their native environments.
Traditional microscopes must collect the transmitting part of the illumination light beam and then construct a two-dimensional image based upon the loss of photons. Traditional microscopes cannot image tiny particles with lateral sizes under one half of the illumination wavelength because of the diffraction limit. However, laser scattering could illuminate particles well below this limit, down to 10 nanometers, and still be detected by traditional charge-coupled devices (CCDs).
Traditional dark-field microscopy has already demonstrated the ability to see dense particles under optical resolutions. Dark-field microscopy generally relies on broadband light sources for illumination of a sample. However, the availability of lasers, which have well-defined properties and are easily manipulated in terms of wavelength, direction, and intensity, has led to the substitution of broadband light sources with lasers in dark-field microscopy. Current developments with laser-scattering microscopes have already demonstrated the ability to see organic particles down to 20 nanometers (nm) and metallic particles down to 5 nm. Additional research has demonstrated that motion of such particles could be resolved to 10 nm or better when using a pin hole filter after the focal spot at the imaging plane.
Scanning confocal microscopes provide three-dimensional images, but only for areas of a sample tainted with fluorescent dye before imaging. However, even the scanning confocal microscope does not reveal optical or other properties of the surfaces which do not have fluorophores. Typical dark-field microscopes are also limited in that they do not provide detailed illumination information; certainly not enough to construct a three-dimensional image.
Scanning Tunneling Microscopy (STM) and Near-Field Scanning Optical Microscopy (NSOM) also only provide information on the surface of a sample, although they have much better resolution than traditional microscopes.
Thus, a need exists in the art for a dark-field laser-scattering microscope which is capable of analyzing single macromolecules in their in-vivo environment and capable of determining exact size, shape, molecular weight, and refractive index.